Differentiation of pluripotent cells into microglial cells expressing IBA-1

ABSTRACT

The present invention relates to a method of culturing primitive macrophages from stem cells. Specifically, the method comprises contacting and incubating stem cells with a serum-free culture media comprising a GSK3 inhibitor to differentiate stem cells into cell of the mesoderm lineage, contacting and incubating cells of the mesoderm lineage with a culture media comprising DKK1 to differentiate the cells into the hematopoietic lineage, maturing the cells of the hematopoietic lineage and contacting and incubating these cells with a culture media comprising M-CSF to drive differentiation into primitive-like macrophages. The invention also relates to a primitive-like macrophage, use of the primitive-like macrophage and a kit when used in the method of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional application of a U.S. patentapplication Ser. No. 15/544,254, filed on Jul. 17, 2017, which is a U.S.National Phase application under 35 U.S.C. § 371 of InternationalApplication No. PCT/SG2016/050018, filed on Jan. 18, 2016, entitledDIFFERENTIATION OF MACROPHAGES FROM PLURIPOTENT STEM CELLS, which claimsthe benefit of priority of Singapore application number 10201500366V,filed Jan. 16, 2015, the contents of which were hereby incorporated byreference in the entirety for all purposes.

FIELD OF THE INVENTION

The invention relates to a differentiation of macrophages from stemcells. Specifically, the invention relates to a serum-free, feeder-freeand embryoid body-free method for differentiation of primitivemacrophages from stem cells.

BACKGROUND OF THE INVENTION

Macrophages are mononuclear phagocytes that play a crucial role intissue homeostasis and immunity, but also contribute to a broad spectrumof pathologies and thus represent key therapeutic targets. However, indepth insights into the differentiation of primitive macrophages fromhuman pluripotent stem cells, their homeostasis, as well as theirdefined activities in a tissue-specific context are currently lacking.

According to the mononuclear phagocyte system (MPS) concept, homeostasisof tissue resident macrophages relies on the constant recruitment ofblood monocytes. However, while monocytes clearly give rise tomacrophages in pathological settings and inflammation, recent evidencehas shown that (1) monocytes do not substantially contribute to certaintissue macrophages under steady state and in certain types ofinflammation, and (2) some adult tissue macrophages such as brainmicroglia are derived from embryonic primitive precursors calledprimitive macrophages that seed tissues prior to birth and maintainthemselves in adults by self-renewal.

Based on this evidence, there is a need to recapitulate the embryonicprimitive origin of macrophages in vitro in order to design new clinicalstrategies targeted to monocytes and macrophages. In this regard, thevast majority of existing approaches to hematopoietic differentiation ofhuman cells have utilized either co-culture with a stromal layer, or asembryoid bodies (EBs). Many of these protocols rely on the use of anundefined bovine serum supplement. These conditions introduce intrinsiclevels of variability into protocols for hematopoietic differentiationof human cells that can affect reproducibility and yields. Accordingly,there is a need to provide a method for hematopoietic differentiationthat overcomes, or at least ameliorates, one or more of thedisadvantages described above to recapitulate the embryonic origin ofmacrophages in vitro.

SUMMARY OF THE INVENTION

In one aspect, there is provided a method for culturing primitive-likemacrophages from stem cells wherein the method comprises:

(a) contacting and incubating said stem cells with a serum-free culturemedia comprising a GSK3 inhibitor to induce differentiation of said stemcells into cells of the mesoderm lineage;

(b) contacting and incubating said cells of the mesoderm lineage with aculture media comprising DKK1 to differentiate the cells of the mesodermlineage into cells of the hematopoietic cell lineage;

(c) maturing said cells of the hematopoietic cell lineage;

(d) contacting and incubating said mature cells of the hematopoieticcell lineage with a culture media comprising M-CSF to drive thedifferentiation of said hematopoietic cells into primitive-likemacrophages.

In one aspect, there is provided a kit when used in the method asdefined herein, comprising a GSK3 inhibitor and DKK1 with instructionsfor use.

In one aspect, there is provided a primitive-like macrophage obtained bythe method as described herein.

In one aspect, there is provided a use of the primitive-like macrophageas described herein for developing in vitro disease models, wherein thedisease is a neurodegenerative disease, a metabolic disease, arespiratory disease, a cardiovascular disease, a connective tissuedisease, cancer or an inflammatory disease.

In one aspect, there is provided a use of the microglial cell asdescribed herein for developing in vitro disease models, wherein thedisease is a neurodegenerative disease.

In one aspect, there is provided a use of the primitive-like macrophageas described herein for screening compounds to treat a disease, whereinthe disease is a neurodegenerative disease or a metabolic disease.

In one aspect, there is provided a use of the microglial cell asdescribed herein for screening compounds to treat a disease, wherein thedisease is a neurodegenerative disease.

In one aspect, there is provided a use of the primitive-like macrophageas described herein for in the manufacture of a medicament for woundhealing and tissue regeneration.

In one aspect, there is provided a use of the primitive-like macrophageas described herein for delivering a cargo molecule into a tissue,wherein the cargo molecule is an immunomodulatory cytokine or chemokine,or an enzyme that activates pro-drugs.

In one aspect, there is provided a method for culturing primitive-likemacrophages from murine stem cells wherein the method comprises:

-   -   (a) contacting and incubating said stem cells with a serum-free        culture media comprising FGF2 and BMP4 to induce differentiation        of said stem cells into cells of the mesoderm lineage;    -   (b) contacting and incubating said cells of the mesoderm lineage        with a culture media comprising FGF2, BMP4, Activin A and VEGF        to differentiate the cells of the mesoderm lineage into cells of        the hematopoietic cell lineage;    -   (c) maturing said cells of the hematopoietic cell lineage;    -   (d) contacting and incubating said mature cells of the        hematopoietic cell lineage with a culture media comprising M-CSF        to drive the differentiation of said hematopoietic cells into        primitive-like macrophages.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detaileddescription when considered in conjunction with the non-limitingexamples and the accompanying drawings, in which:

FIG. 1 shows a flow diagram of the method of the invention.

FIG. 2 shows the timeline for the addition of growth factors used in themethod of the invention.

FIGS. 3A-3C show the flow cytometry analysis of macrophagesdifferentiated from H1 ES cells (FIG. 3A), patient-derived WT iPSCs(FIG. 3B) and patient-derived mutant iPSCs (FIG. 3C): Cells aresequentially gated on their FSC/SSC profile, then live cells (DAPI−),then hematopoietic cell (CD45+) and then singlets (SSC-A and SSC-Wgate). Profile of expression of common macrophage markers are shown:CD14, HLA-DR, CD11b and CD163. Macrophages differentiated from H1 EScells, patient derived WT iPSCs and patient-derived mutant iPSCs areCD14+, HLA-DR+ and most of them CD163+ and CD11b+. (SSC=side scatter:Side scatter measures scattered light at 90 degrees to the laser pathand measures the granularity of the cell; FSC=Forward Scatter: Forwardscatter measures scattered light in the direction of the laser path andmeasures the size of the cell; DAPI (4′,6-Diamidino-2-phenylindoledihydrochloride): CD45: Pan hematopoietic cell marker; HLA-DR:Macrophage and dendritic cell marker, MHC class II cell surface receptorencoded by the human leukocyte antigen complex; CD14: cluster ofdifferentiation 14, Macrophage marker; CD11b: Integrin alpha M (ITGAM),Macrophage marker; CD163: cluster of differentiation 163, high affinityscavenger receptor for the hemoglobin-haptoglobin complex, Macrophagemarker).

FIG. 4 shows the results of phagocytosis assays on day 12-sortediPSCs-derived primitive macrophages when exposed to fluorescentlylabeled latex beads or amyloid Aβ peptides. IBA-1=ionizedcalcium-binding adapter molecule 1 (Microglia and macrophage marker);CX3CR1=fractalkine receptor; DIC=Differential interference contrast.

FIG. 5 shows microscope images of the results from the co-culture ofiPSC-derived primitive macrophages on neurons after 7 days of culture.Tuj1=neuron-specific class III beta-tubulin marker; Iba1=ionizedcalcium-binding adapter molecule 1 (Microglia and macrophage marker);Hoechst=Fluorescent dye that labels DNA and then the nucleus of cells.The number in the top right of the images (*60 and *100) indicatemicroscope magnification.

FIG. 6 shows microscope images of the results from the co-culture ofiPSC-derived primitive macrophages on iPSC-derived neurons (Same sourceof iPSC) at different days (7, 16 and 21) of co-culture.

FIG. 7 shows an illustration of the strategy of transferring murineiPSC-derived primitive macrophages intranasally on the CSF2aR-KO Mouse(CD45.2) model in order to test their function.

FIGS. 8A-8C show the flow cytometry analysis of a wild type (WT) Mouse'slung (8-month-old) (FIG. 8A); CSF2aR-KO Mouse lung (9-month-old) (FIG.8B), where no alveolar macrophages are shown (differentiation defect);and CSF2aR-KO Mouse's lung (1.5-month-old) (FIG. 8C)+Positive Control(Frozen iPSC Macrophage).

FIG. 9 shows the flow cytometry analysis of a CSF2aR-KO Mouse's lung(9-month-old) grafted with iPSC Mφ (1000K) for 42 Days. My refers toMacrophage.

FIG. 10 shows that murine iPSC-derived primitive macrophages are able toengraft into the lung for more than 8 weeks and acquire markers ofalveolar macrophages (SiglecF).

FIG. 11 shows that murine iPSC-derived primitive macrophages(CX3CR1-GFP+) may engraft into the lung for more than 8 weeks andphagocyte the Surfactant Associated Protein B (SPB).

FIG. 12 shows the turbidity of the bronchoalveolar lavage fluidcollected from CSF2aR-KO Mice (KO) and CSF2aR-KO Mice-grafted withiPSC-derived primitive macrophages (KO-graft). The optical density ofthe lavage fluid was measured at 600 nm.

FIG. 13 shows the turbidity of the bronchoalveolar lavage fluidcollected from wild type mouse (WT), CSF2aR-KO Mice (KO) and CSF2aR-KOMice-grafted with iPSC-derived primitive macrophages (KO-graft). Theoptical density of the lavage fluid was measured at 600 nm.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In a first aspect the present invention refers to a method for culturingprimitive-like macrophages from stem cells. The method comprises: (a)contacting and incubating said stem cells with a serum-free culturemedia comprising a GSK3 inhibitor to induce differentiation of said stemcells into cells of the mesoderm lineage; (b) contacting and incubatingsaid cells of the mesoderm lineage with a culture media comprising DKK1to differentiate the cells of the mesoderm lineage into cells of thehematopoietic cell lineage; (c) maturing said cells of the hematopoieticcell lineage; and (d) contacting and incubating said mature cells of thehematopoietic cell lineage with a culture media comprising M-CSF todrive the differentiation of said hematopoietic cells intoprimitive-like macrophages.

The stem cells may be selected from embryonic stem cells (ESC) andinduced pluripotent stem cells (iPSC). In one embodiment the stem cellsmay be human H1 embryonic stem cells (ESC). In another embodiment thestem cells may be human induced pluripotent stem cells (iPSC).

The GSK3 inhibitor may be selected from 6-bromoindirubin-3′-oxime (BIO),CHIR-99021, SB216763, CHIR-98014, TWS119, IM-12, 1-Azakenpaullone,AR-A014418, SB415286, AZD1080, AZD2858, indirubin, A 1070722, TCS 2002,Tideglusib, or any derivatives thereof.

In some embodiments the stem cells may be plated at a density of lessthan 1 small colony/cm² on Matrigel-coated 6-well plates at least 24hours prior to step (a).

The stem cells may be incubated with a media comprising 80% DMEM/F12,20% knockout serum replacement, L-glutamine, non-essential amino acids,beta-mercaptoethanol and FGF-2, step (a). In some embodiments, the stemcells may be incubated for about 1 day in said media.

In another embodiment, the stem cells may be contacted at step (a) witha differentiation media, comprising 1-20 ng/ml of BMP4, 5-100 ng/ml ofVEGF, 0-50 ng/ml of FGF-2, and 0-10 μM of CHIR99021. The differentiationmedia may be StemPro® 34 from Invitrogen. The cells may be incubatedwith the media for about 2 days in an oxygen concentration of about 5%.

In another embodiment, the cells are further contacted at step (a) with1-20 ng/ml of BMP4 and 5-100 ng/ml of VEGF.

In another embodiment, the cells are incubated for about 2 days withsaid media in an oxygen concentration of about 5%.

The cells may be contacted at step (b) with a differentiation mediacomprising 5-100 ng/ml of VEGF; 0-50 ng/ml of FGF-2; 0-250 ng/ml of SCF;0-500 ng/ml DKK1; 0-50 ng/ml of IL-6; and 0-50 ng/ml of IL-3. The cellsmay be incubated for about 3 days with said media in an oxygenconcentration of about 5%.

In some embodiments, the cells at step (c) may be matured by contactwith a differentiation media comprising 0-50 ng/ml of FGF-2; 0-250 ng/mlof SCF; 0-50 ng/ml of IL-3 and 0-50 ng/ml of IL-6. The cells may beincubated with the media for about 7 days in normoxic conditions. Theterm “normoxic conditions” as used herein refers to conditions where theoxygen concentration levels may be about 20% in the media.

The cells may be contacted and incubated at step (d) with adifferentiation media comprising 0-100 ng/ml M-CSF. The cells may beincubated with the media for at least 6 days in normoxic conditions.

In one embodiment the method as described herein may produce a yield ofprimitive-like macrophages of greater than 1×10⁶/well in a 6-wellculture plate between 21 to 25 days of culture. As will be appreciatedin the art, the yield of the macrophages may be dependent upon theorigin of the cells that are applied in the methods described herein.

In another embodiment the method may further comprise isolating saidprimitive-like macrophages using FACS sorting or magnetic separation.

Also provided herein is a kit when used in accordance with the method asdescribed herein, comprising a GSK3 inhibitor and DKK1 with instructionsfor use.

Also provided herein is a primitive-like macrophage obtained by themethod as described herein. The primitive-like macrophage may besubsequently differentiated into a microglial cell, an alveolarmacrophage, Kupffer cell, Langerhans cell, or other tissue macrophages.The term “macrophages of other tissues” refers to macrophages that maybe found in any tissue type including but not limited to kidney tissue,pancreas tissue, adipose tissue, liver tissue or connective tissue.

Also provided herein is the use of the primitive-like macrophage asdescribed herein for developing in vitro disease models, wherein thedisease is a neurodegenerative disease, a metabolic disease, arespiratory disease, a cardiovascular disease, a connective tissuedisease, cancer or an inflammatory disease. In one embodiment thedisease is a neurodegenerative disease.

In some embodiments, there is provided use of the primitive-likemacrophage as described herein for screening compounds to treat adisease, wherein the disease is a neurodegenerative disease or ametabolic disease.

In some embodiments, the disease is a neurodegenerative disease. In someembodiments, the neurodegenerative disease may be Alzheimer's disease,Huntington's disease or Rett syndrome. In some embodiments, themetabolic disease may be obesity or diabetes. In some embodiments, therespiratory disease may be pulmonary alveolar proteinosis. In someembodiments, the cardiovascular disease may be atherosclerosis. In someembodiments, the connective tissue disease may be fibrosis. In someembodiments the inflammatory disease may be arthritis, rheumatoidarthritis, experimental autoimmune encephalomyelitis, multiple sclerosisor inflammatory bowel disease as well as skin inflammatory disease suchas Atopic Dermatitis, Psoriasis.

There is also provided the use of the primitive-like macrophage asdescribed herein for wound healing and tissue regeneration.

There is also provided the use of the primitive-like macrophage asdescribed herein for delivering a cargo molecule into a tissue, whereinthe cargo molecule is an immunomodulatory cytokine or chemokine, or anenzyme that activates pro-drugs.

There is also provided a method for culturing primitive-like macrophagesfrom murine stem cells wherein the method comprises: (a) contacting andincubating said stem cells with a serum-free culture media comprisingFGF2 and BMP4 to induce differentiation of said stem cells into cells ofthe mesoderm lineage; (b) contacting and incubating said cells of themesoderm lineage with a culture media comprising FGF2, BMP4, Activin Aand VEGF to differentiate the cells of the mesoderm lineage into cellsof the hematopoietic cell lineage; (c) maturing said cells of thehematopoietic cell lineage; (d) contacting and incubating said maturecells of the hematopoietic cell lineage with a culture media comprisingM-CSF to drive the differentiation of said hematopoietic cells intoprimitive-like macrophages.

The murine stem cells may be selected from mouse embryonic stem cells(ESC) and mouse induced pluripotent stem cells (iPSC).

The invention illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims and non-limitingexamples. In addition, where features or aspects of the invention aredescribed in terms of Markush groups, those skilled in the art willrecognize that the invention is also thereby described in terms of anyindividual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION

Non-limiting examples of the invention, including the best mode, and acomparative example will be further described in greater detail byreference to specific Examples, which should not be construed as in anyway limiting the scope of the invention.

Example 1

Differentiation of Mouse Pluripotent Stem Cells into PrimitiveMacrophages

If mESC/miPSC are expanded on MEF, deplete MEF by 1-2× passage ongelatin-coated 6-well plates before commencing with differentiation.

On day 0, colonies are dissociated and resuspended at 175,000-200,000cells/ml in serum-free differentiation medium (recipe below)supplemented with 5 ng/ml hFGF2 and 5 ng/ml hBMP4. These are dispensedinto non-adherent plates to allow for embryoid body (EB) formation.

After 48 h, EBs are pooled and dissociated with TrypLE (preferred) orTrypsin.

EBs are then reaggregated at 175,000 cells/ml in serum-freedifferentiation medium supplemented with 5 ng/ml hFGF2, 2 ng/ml hBMP4, 2ng/ml human Activin A, 5 ng/ml hVEGF.

After a further 48 h, EBs are dissociated and sorted for Flk-1+ cells.

Sorted cells are then reaggregated at 500,000 cells/ml in serum-freedifferentiation medium supplemented with 5 ng/ml VEGF, 300 ng/ml DKK1,100 ng/ml M-CSF. The cell suspension is then dispensed into non-adherentculture wells to allow EB formation.

After a further 48 h (d6 of differentiation total), EBs are dissociatedand resuspended in StemPro SFM complete medium (recipe below)supplemented with 100 ng/ml SCF, 10 ng/ml IL-3, 100 ng/ml M-CSF. Thecells are then plated onto gelatin-coated tissue culture plates.

Primitive-like macrophages should emerge starting from d8 ofdifferentiation and peak around d13-14 of differentiation. Typicallycells are sorted or harvested around d10-11. It is not necessary topassage the cells, but note that there will be many cells in suspensionby d8. Collect both suspension and adherent fractions for sorting.

Serum-Free Differentiation Medium:

-   75% IMDM/25% F12-   0.5×N2 supplement-   0.5×B27 supplement (without retinoic acid)-   1× pen/strep-   0.05% BSA-   2 mM L-glutamine-   0.5 mM ascorbic acid-   4.5×10⁻⁴ M 1-thioglycerol

StemPro SFM Complete Medium:

-   StemPro-34 SFM (Invitrogen)-   1× pen/strep-   2 mM L-glutamine-   200 ug/ml transferrin-   0.5 mM ascorbic acid-   4.5×10⁻⁴ M 1-thioglycerol

Example 2

Differentiation of Human Pluripotent Stem Cells into PrimitiveMacrophages

The differentiation strategy can be divided into four stages: primitivestreak/mesoderm induction, hematopoietic specification, hematopoieticcell maturation, and finally terminal myeloid expansion. Theexperimental details of each stage are illustrated in FIGS. 1 and 2 ,and described below:

Stage 0: ES or iPS cells are maintained on Matrigel-coated dishes in CF1MEF-conditioned media. The media recipe consists of 80% DMEM/F12, 20%knockout serum replacement, L-glutamine, non-essential amino acids,beta-mercaptoethanol, and 6 ng/ml FGF-2 and media is replaced daily.Cells are passaged every 5-6 days using collagenase IV treatment andgentle detachment with a cell scraper. 24 hours prior to initiation ofdifferentiation, cells are plated at a very low density (<1 smallcolony/cm² on Matrigel-coated 6-well plates and allowed to attachovernight.

Stage 1 (primitive streak/mesoderm induction): In this initial stage,cells are treated with BMP4 and GSK3 inhibitor to promote specificationto the posterior primitive streak, which gives rise to the mesodermduring embryogenesis. VEGF is also added to promote the development ofhematopoietic mesoderm. The cells are also maintained under hypoxicconditions (5% O₂).

Stage 2 (hematopoietic specification): After approximately 4-5 days, asubset of cells with high expression of KDR and CD31 can be observed.This population contains the hemangioblast, the bipotential precursor tohematopoietic and endothelial cells. The induction cocktail at thispoint is tailored to expand hemangioblast cells and further specify themtowards the hematopoietic lineage. Here, SCF and FGF-2 are added topromote cell viability and proliferation. IL-3, IL-6 and VEGF are addedto promote hematopoietic differentiation, and DKK1 is added to suppresserythroid differentiation (and perhaps drive macrophagedifferentiation). Cells are maintained in hypoxic conditions until day8, after which they are cultured in normoxia.

Stage 3 (Hematopoietic cell maturation): In this stage, hematopoieticcells are driven more fully towards the macrophage lineage. DKK and VEGFare no longer necessary by this point and are thus removed from thecocktail.

Stage 4 (terminal myeloid expansion): All cytokines are removed with theexception of M-CSF, which was found to promote the proliferation ofprimitive macrophages via signaling through the CSF1-receptor.

Macrophages expressing classical surface markers can be observed in thepopulations as early as day 18 of differentiation, and we have foundthat flow sorting of cells between day 21-25 of differentiation resultsin good yield and viability. Specifically, the expression profile ofcommon macrophage markers (CD14, HLA-DR, CD11b and CD163) are shown inFIG. 3 with respect to macrophages differentiated from H1 ES cells,patient derived WT iPSCs and patient-derived mutant iPSCs.

Differentiation Medium 1 (DM1)

-   StemPro 34 (Invitrogen) with the supplement and 1× pen/strep-   2 mM glutamine-   1× apo-transferrin-   4×10⁻⁴M monothioglycerol (MTG)-   50 ug/ml ascorbic acid (AA)

Differentiation Medium 2 (DM2)

-   IMDM 75%/Ham's F-12 25% with 1× pen/strep-   0.5×B27 supplement (without retinoic acid)-   0.5×N2 supplement-   2 mM glutamine-   4×10⁻⁴M monothioglycerol-   50 ug/ml ascorbic acid

Procedure

-   Day 0: Exchange expansion media with DM1 containing BMP4 (5 ng/ml),    VEGF (50 ng/ml), CHIR99021 (2 μM). Culture in 5% 02.-   Day 2: Exchange media with DM1 containing BMP4 (5 ng/ml), VEGF (50    ng/ml), FGF-2 (20 ng/ml). Culture in 5% O₂.-   Day 4: Exchange media with DM1 containing VEGF (15 ng/ml), FGF-2 (5    ng/ml). Culture in 5% O₂.-   Day 6: Exchange media with DM1 containing VEGF (10 ng/ml), FGF-2 (10    ng/ml), SCF (50 ng/ml), DKK1 (30 ng/ml), IL-6 (10 ng/ml), IL-3 (20    ng/ml). Culture in 5% O₂.-   Day 8, 10: Exchange media with DM1 containing VEGF (10 ng/ml), FGF-2    (10 ng/ml), SCF (50 ng/ml), DKK1 (30 ng/ml), IL-6 (10 ng/ml), IL-3    (20 ng/ml). Return cells to normoxic conditions (20% O₂).-   Day 12, 14: Exchange media with DM1 containing FGF-2 (10 ng/ml), SCF    (50 ng/ml), IL-6 (10 ng/ml), IL-3 (20 ng/ml).-   Day 16, 18, 20: Exchange media with DM2 containing MCSF (50 ng/ml).

Technical Notes

A volume of 3 ml media/well in the 6 well plate is recommended.

From day 6 onwards, cells in suspension are observed. To avoid loss ofhematopoietic cells, the supernatant is centrifuged and the resultantcell pellet is re-suspended in a small quantity of fresh media beforebeing added back to their original wells.

DM1 should be freshly prepared every 5-7 days and stored at 4° C. tominimize loss of bioactivity.

DM2 can be prepared (without AA and MTG) in advance and frozen inaliquots before use. AA and MTG can be added to thawed aliquots andstored for up to one week at 4° C.

There are a number of unique and advantageous features of this methodincluding that it mimics primitive hematopoiesis in the embryonic yolksac. This method is completely serum- and feeder-free, as compared tocompeting technologies that require serum supplementation later on andthat require co-culture with stromal cells such as OP9. In addition, nodissociation steps are required and there is no embryoid body formationstage. Rather, differentiation takes place on 2D adherent culture overthe entire process. The method has also been validated on H1 cells andother iPSC cell lines and is scalable, resulting in high yield ofprimitive macrophages (>1×10⁶/well in a 6 well plate). The addition ofthe GSK inhibitor pushes initial specification towards the primitivestreak while the addition of DKK suppresses erythroid differentiation.

Example 3

Differentiation of Human iPSC-Derived Primitive Macrophages

Phagocytosis assays were performed with day 12-sorted iPSCs-derivedprimitive macrophages (CD163+CD11b+HLA-DR+IBA-1+CX3CR1+CD14+), whereinthe macrophages have been differentiated from patient derived WT iPSCsand the expression profile is CD45+CD11b+, and CD163+. Specifically, theday 12-sorted iPSCs-derived primitive macrophages were exposed tofluorescently labeled latex beads or amyloid Aβ peptides in order toassess their capacity to phagocyte as macrophages and microglia.

As shown in FIG. 4 , the capacity of said iPSC-derived primitivemacrophages to phagocyte as macrophages and microglia is supported. Thisis of importance since amyloid beta (Aβ or Abeta) peptides are cruciallyinvolved in Alzheimer's disease as the main component of the amyloidplaques found in the brains of Alzheimer patients.

Procedure

The method for the analysis of macrophage phagocytic ability used acontrol line of Huntington's disease human iPSCs (HD33i) derivedprimitive macrophages that were reconstituted with IMDM containing 10%FCS, seeded (5.0×10⁴ cells/well) on the 24-well-dish, and settledovernight at 37° C. in a fully humidified atmosphere of 5% CO2 in air.

Cells were then incubated with latex beads (1:100,000) for 24 hours,washed with PBS three times, fixed with 4% PFA, and analyzed by laserconfocal microscopy.

Phagocytosed latex beads were analyzed by the reference of cellmorphology (differential interference contrast) and nuclei (DAPI,1:10,000).

Example 4

Human iPSC Derived Neuron and Macrophage Co-Culture.

iPSC-derived primitive macrophages obtained from the methods describedherein were co-cultured with neurons.

As shown in FIGS. 5 and 6 , the co-culture led to the differentiation ofthe iPSC-derived primitive macrophages into IBA1+ microglia with aramified morphology reminiscent of in vivo adult microglia.

These results show that iPSC-derived primitive macrophages have thecapacity to differentiate into microglial cells.

Procedure

Differentiation methods described herein were adopted to induce theneurons from human iPSCs (HD33i). Eighty percent (80%) confluency ofiPSCs were washed with DMEM/F12 two times and dissociated to singlecells with accutase. Cells were re-suspended with neuron precursor cell(NPC)+ medium and plated on the matrigel coated 6-well-dish. NPC+ mediumwas changed every day for 7 days for the induction of NPCs. NPCs werethen split at 1:3 dilution in NPC− medium with 10 μM ROCK inhibitor onthe matrigel coated 6-well-dish and followed by several passages for theconditioning with NPC− media supplemented with EGF (20 ng/ml) and bFGF(20 ng/ml). For the terminal differentiation to neurons, confluent NPCswere dissociated to single cells with accutase, re-suspended with neurondifferentiation medium, and seeded (5.0×10⁴ cells/well) on the coverslipcoated with poly-L-ornithine and laminin in 24-well-dish.

Example 5

CSF2aR-KO Mice Lung-Transplantation

A CSF2aR-KO Mice (CD45.2) mouse model of Pulmonary Alveolar Proteinosiswas used in testing the effect of iPSC-derived primitive macrophages.

As shown in FIG. 7 , the transfer of murine iPSC-derived primitivemacrophages rescued and alleviated the Pulmonary Alveolar Proteinosisdisease in the mouse model.

Cell suspensions from the lung of non-treated wild type mouse (8 a,positive control), the non-treated mouse model CSF2aR-KO (8 b, negativecontrol) and the mouse model CSF2aR-KO with added iPSC macrophages inthe cell suspension as a technical control (8 c) were analysed throughflow cytometry and the presence or absence of alveolar macrophagesdetected through the visualisation of known macrophage markers.

In this regard, the circled areas in FIGS. 8 a, b and c show thepresence or absence of alveolar macrophages, wherein alveolarmacrophages are present in FIG. 8 a and absent in FIGS. 8 b and 8 c .FIGS. 8 a, b and c demonstrate that prior to intranasal transfer of exvivo-derived macrophages, CSF2aR-KO mice are deficient in alveolarmacrophages.

Moreover, in a further experiment the CSF2ar-KO mouse receivedintranasal transfer of 1000K iPSC CX3CR1-GFP macrophages, and cellsuspensions of the lung were analysed by flow cytometry. As shown inFIGS. 9 and 10 , murine iPSC-derived primitive macrophages (iPSC derivedmacrophages are F4/80 and CX3CR1-GFP and acquire Siglec-F expression)engraft into the lung for more than 42 days and acquire markers ofalveolar macrophages (SiglecF) while concurrently maintaining theexpression of specific markers such as CD11b. Specifically, the dottedcircle in FIG. 9 represents the expected population of endogenousalveolar macrophages present in WT mouse.

Murine iPSC-derived primitive macrophages are also shown to engraft intothe lung for more than 8 weeks and acquire markers of alveolarmacrophages (SiglecF) and distribute into the whole lung (FIG. 10 ).

Murine iPSC-derived primitive macrophages (CX3CR1-GFP+) were also shownto engraft into the lung for more than 8 weeks and phagocyte theSurfactant Associated Protein B (SPB) accumulated due to the absence ofendogenous functional alveolar macrophages (See FIGS. 10 and 11 ).Consequently, there is less accumulation of SPB in the grafted lung andthe turbidity of the bronchoalveolar measured by ELISA lavage is reduced(FIGS. 12 and 13 ). As will be appreciated, the effects and resultsshown on the CSF2aR-KO Mice (CD45.2) mouse model are a sign that thedisease is cleared as a consequence of the treatment with iPSC-derivedprimitive macrophages.

What is claimed is:
 1. A method for producing microglial cells, themethod comprising: (i) (a) culturing human pluripotent cells in aserum-free culture medium comprising VEGF, BMP4 and a GSK3 inhibitorunder normoxic conditions such that said pluripotent cells differentiateinto cells of the mesoderm lineage; (b) culturing said cells of themesoderm lineage under hypoxic conditions in a culture medium comprisingSCF, FGF-2, IL-3, IL-6, VEGF and DKK1 such that the cells of themesoderm lineage differentiate into cells of the hematopoietic celllineage; (c) culturing said cells of the hematopoietic cell lineage in aculture media comprising SCF, FGF-2, IL-3 and IL-6 under normoxicconditions such that the cells of the hematopoietic cell lineage mature;(d) culturing said mature cells of the hematopoietic cell lineage in aculture medium comprising M-CSF under normoxic conditions such that saidmature cells of the hematopoietic cell lineage differentiate intoprimitive macrophages that express CD163, CD1 lb, HLA-DR, IBA-1, CX3CR1and CD14; (ii) (a) culturing human pluripotent cells in neuralprogenitor cell (NPC) medium for up to 7 days such that neural precursorcells are obtained; (b) culturing the neural precursor cells in NPCmedium comprising a Rho-associated, coiled-coil containing proteinkinase (ROCK) inhibitor; (c) culturing the cells obtained in step(ii)(b) in NPC medium comprising epidermal growth factor (EGF) and basicfibroblast growth factor (bFGF); (d) culturing the cells obtained instep (ii)(c) in neuronal differentiation medium such that neurons areobtained; and (iii) culturing the primitive macrophages obtained in step(i)(d) with the neurons obtained in step (ii)(d) for up to 21 days suchthat microglial cells expressing ionized calcium binding adaptormolecule 1 (IBA1) are obtained.
 2. The method of claim 1, wherein theNPC medium in step (ii)(b) comprises 10 μM ROCK inhibitor.
 3. The methodof claim 1, wherein the NPC medium in step (ii)(c) comprises 20 ng/mlEGF and 20 ng/ml bFGF.